A vaccinia virus mutant which is resistant to the drug aphidicolin (AP) codes for an AP-resistant DNA polymerase. A change from a wild-type (WT) guanine to a thymine at position 2430 of the sequence of the DNA polymerase gene changes a leucine to a methionine in the polymerase and confers AP-resistance. The mutation was confirmed by site directed oligonucleotide mutagenesis of the WT DNA. To prepare the polymerase gene for the insertion of other mutations near position 2430 which might confer AP-resistance, a part of the WT gene, the Eco B segment, was cloned in a phagemid. Bgl I and Xho I restriction sites were inserted in the cloned Eco B segment at positions 2412 and 2514, respectively. Double-stranded oligonucleotides with sequences identical to the wild-type DNA except for single base changes can be inserted at the Bgl and Xho sites and the recombinant DNA tested for the ability to confer AP resistance. A phosphonoacetate resistance mutation was inserted at position 1537 to provide a means for selecting vaccinia virus with an altered B segment. Different mutagenic procedures were tested for their efficiency during the course of the work. The Bgl and Xho sites were inserted in the DNA by the Kunkel method which uses a bacterial strain that produces single-stranded template DNA containing uracil. This method, adapted for the phagemid system, had a 5% success rate for the creation of both the Bgl and Xho sites. When normal template DNA without uracil was used, the mutation frequency was tenfold less. The Eckstein method of mutagensis uses the restriction enzyme Nci, which will not cleave duplex DNA when one strand contains phosphorothioate nucleotides, but will nick a normal complementary strand. Initial results indicate a 90% success rate for the creation of the desired mutation. The conditions needed to increase the yield of the final DNA product used for the transformation reaction are under investigation.